Cholesterol is a major lipid component of the mammalian plasma membrane. Abnormal cholesterol levels have been implicated in several diseases, including cardiovascular diseases, Niemman-Pick type C disease, and Alzheimer's disease, among others; however it is not known how the cholesterol concentration is linked to cellular processes. In particular, the direct involvement of cholesterol in cell regulation through specific interactions with cytosolic proteins has not been thoroughly investigated. We have recently discovered that cholesterol specifically interacts with various cytosolic scaffold protein and regulate their diverse cellular signaling activities. This important finding not only demonstrates that cholesterol can directly interact with major cellular regulatory proteins but also offers excellent systems to investigate the direct correlation between membrane cholesterol levels and cellular activities. We also developed a new fluorescence imaging technology for accurate in situ real-time quantification of membrane lipids. Collectively, our discovery of a new class of cholesterol binding proteins and our new quantitative lipid imaging technology provide us a unique and unprecedented opportunity to systematically study the mechanisms by which cholesterol regulates diverse cellular processes. Our main hypothesis is that the local cholesterol level in the plasma membrane serves as an activation threshold for various cellular processes, including Wnt signaling. The primary objective of this proposed research is to investigate the mechanisms underlying diverse cholesterol-mediated cell regulation. Specifically, we propose to (1) determine how cholesterol regulates Wnt signaling through specific interaction with a scaffold protein, Dvl, (2) establish robust and versatile in situ quantitative imaging of cellular cholesterol, and (3) determine how changes in the local cholesterol level in the plasma membrane mediate canonical Wnt signaling.